<2022-05-24 Tue> David biweekly meeting

project updates

Project 4: Reconstitution of endocytic actin network

recent progress

EM
  • currently processing hopefully final set of samples for EM
  • I'm optimistic that I solved the issue of beads moving around during EM processing by:
    • incubating fixed samples on dish overnight instead of 1 hour
    • stringently washing away loosely attached beads in buffer 3 times before light micrsocopy
    • marking regions where beads do not move upon shaking the stage back and forth during light microscopy imaging
  • due to optimus prime bright field not working anymore, I worked out a new imaging scheme on the airyscan
    • pro: whole-slide tile scans work really well
    • con: need to manually register fluorescence and bright field channels, though they get distorted slightly differently from each other so it can't be a perfect overlay
  • example tile scan of +Las17 dish
    • bright field

      2022-05-24_13-25-38_Screenshot from 2022-05-24 13-20-12.png
    • cyan lipid, magenta Sac6-GFP

      2022-05-24_13-25-52_MAX_dish_10x_tile_fluo_postwash-01.czi - dish_10x_tile_fluo_postwash-01.czi %236.jpg
time lapse fluorescence imaging
  • purpose: to help interpret the deformed membranes I've observed by EM in the +Las17 +LatA condition
  • finished collecting +/- LatA time lapses on airyscan
    • 3 biological replicates
    • 1-2 movies per replicate, ~1 hour each
    • ~10 beads per movie
  • worked out strategy for systematically 3D visualizing and reproducibly recording vesiculation events on each bead
    • I do an unbiased screen for all movies by hiding the Sac6 channel and only looking for membrane budding events in the lipid channel
    • so far, analyzed 10-20 beads in each condition and only observed obvious membrane budding in +Las17 -LatA condition
    • however, there are stable membrane tubules/irregularities in all conditions
  • example vesiculation event in +Las17 -LatA condition
  • example membrane irregularity in +Las17 +LatA condition

next steps

  • continue EM processing and imaging
  • continue scoring fluorescence time lapse data

Project 7: Mechanical regulation of CME by protein LLPS

feedback from lab meeting?

Date: \today

Author: Max Ferrin

Email: ferrinm@berkeley.edu

Created: 2022-05-24 Tue 13:42

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